Inhibiting the oncogene c-Myc and mutant KRas remains a major challenge in cancer research. c-Myc and KRas are hyperactive in more than 70% of all human cancers, and contribute to resistance to therapies. However, drugs that directly inhibit c-Myc or mutant KRas have not yet been developed. c- Myc and KRas are key oncogenic drivers in colorectal cancer, a leading cause of cancer death; nearly 100% of colorectal cancers depend on c-Myc, while KRas mutations occur in ~50% of colorectal cancers. Recent findings indicate that post-translational modification by the Small Ubiquitin-like MOdifiers (SUMO) play a critical role in c-Myc and KRas-dependent oncogenesis; however, the mechanisms of how SUMOylaton is involved in c-Myc and KRas-dependent oncogenesis are not well understood. Wnt-signaling pathways could be a critical link of SUMOylation to both c-Myc activation and KRas mutation in colorectal cancers. Dysregulated Wnt-signaling leads to increased c-Myc expression and activity. In addition, KRas mutation suppresses the non-canonical Wnt signaling pathway, thereby enhancing the canonical Wnt-signaling pathway. Wnt signaling is also key to maintaining the pool of cancer stem cells in a wide range of cancers. During the previous funding period, we found that inhibition of SUMOylation significantly suppressed Wnt down-stream targets, including c-Myc. Key proteins, both in the canonical and non-canonical Wnt pathways, have been recently shown to be substrates of SUMOylation. However, the current understanding of how SUMOylation regulates the Wnt pathways is incomplete, and thus will be investigated here using colorectal cancer as a model system. We will conduct a combination of structural studies and chemical synthesis to determine the mechanism of allosteric inhibition by our lead small molecule SUMOylation inhibitor. We will conduct biochemical and molecular biological studies to elucidate the mechanism of SUMOylation in the Wnt signaling pathways. Finally, we will determine the therapeutic efficacy of our lead SUMOylation small molecule inhibitor in c-Myc and KRas-dependent colorectal cancers. We expect that the proposed studies will lead to a new paradigm by providing proof of principle for much-needed therapies that target c-Myc and KRas and dysregulated Wnt-signaling. Our work will also have implications for the many other cancers that are dependent on c-Myc and KRas. !